Methods of using pomegranate extracts for causing regression in lesions due to arteriosclerosis in humans

ABSTRACT

Methods of using pomegranate extracts of the present invention for treating patients with atherosclerosis, or increased intima-media thickness of an artery, are provided. The methods comprise the step of administering to the patient a composition comprising a therapeutically effective amount of an extract from pomegranate. The methods of the present invention may also be used to decrease the incidence of stroke or heart attack in a patient.

RELATED APPLICATION

This is a continuation-in-part of the U.S. application Ser. No.09/294,307, filed on Apr. 19, 1999, now U.S. Pat. No. 6,387,418, thecontent of which is incorporated herein in its entirety by reference.This application also claims priority to the U.S. provisionalapplication serial No. 60/318,160, filed on Sep. 6, 2001, under 35U.S.C. 119(e), the content of which is incorporated herein in itsentirety by reference.

BACKGROUND OF THE INVENTION

1. Area of the Art

The invention relates generally to pomegranate extracts and methods ofusing thereof, and specifically to methods of using pomegranate extractsfor causing regression in lesions due to arteriosclerosis in humans.

2. Description of the Prior Art

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims.

Oxidative stress, a major contributor to cardiovascular diseases (1), isassociated with lipid peroxidation in arterial macrophages and inlipoproteins (1-3). Oxidized low-density lipoprotein (Ox-LDL) was shownto be atherogenic (2-4), thus, interventions to inhibit LDL oxidation bydietary antioxidants (4, 5) is of major importance to attenuateatherosclerosis. It was recently shown that vitamin E supplementation topatients with carotid artery stenosis inhibited LDL accumulation inarterial macrophages (6). Protection of lipids from oxidation can bealso achieved by serum paraoxonase (PON1, an HDL-associated esterasethat can hydrolyze and reduce specific lipid peroxides in arterial cellsand lipoproteins in coronary and carotid lesions (7-10).

The pomegranate was recently chosen as the logo for the MillenniumFestival of Medicine, mainly because of its medicinal properties asdescribed by all major religions and by folk medicine (11). Pomegranatejuice (PJ) possesses impressive antioxidative properties due to its highflavonoids content, mainly the water soluble tannins and proanthocyanins(12). We have recently shown the antioxidative and antiatherogeniccharacteristics of PJ consumption in atherosclerotic apolipoprotein Edeficient (E^(o)) mice (13). In healthy humans, PJ consumption alsodemonstrated potent antioxidative capabilities against lipoproteinoxidation, and also increased PON1 activity and improved serum totalantioxidant status (13).

A need exists, however, to study whether the above beneficial effects ofPJ can be manifested in patients with atherosclerosis such as carotidartery stenosis.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discovery thatpomegranate juice consumption by a patient with atherosclerosis maycause regression of the size of atherosclerotic lesions. Prior to thepresent invention, it has been observed that pomegranate juiceconsumption by E⁰ mice may slow down the progression ofarteriosclerosis, and reduce the expected increase in the size ofatherosclerotic lesions. It is the discovery of the present inventionthat pomegranate juice consumption by a patient with atherosclerosis,however, not only can slow down the progression of arteriosclerosis, butalso can reduce the size of atherosclerotic lesions that are alreadydeveloped, and therefore cause regression of the lesion size.

Accordingly, one aspect of the present invention provides a method fortreating a patient with arteriosclerosis. The method comprises the stepof administering to the patient a composition comprising atherapeutically effective amount of an extract from pomegranate fruit.According to embodiments of the present invention, the arteriosclerosismay be carotid artery arteriosclerosis or coronary arteryarteriosclerosis. The extract of pomegranate may be a juice extract ofpomegranate, an extract from inner or outer peel of pomegranate, or themixture thereof.

Another aspect of the present invention provides a method of treating apatient with an increased intima-media thickness of an artery. Themethod comprises the step of administering to the patient a compositioncomprising an amount of an extract from pomegranate which istherapeutically effective to reduce the intima-media thickness of theartery. According to embodiments of the present invention, theintima-media thickness of the artery is due to arteriosclerosis, suchas, but not limited to, coronary artery arteriosclerosis and carotidartery stenosis, diabetes, high blood pressure, and peripheral vasculardisease.

A further aspect of the present invention provides a method ofdecreasing the incidence of stroke and heart attack in a patient. Themethod comprises the step of administering to the patient a compositioncomprising a therapeutically effective amount of an extract frompomegranate. According to the embodiments of the present invention, thestroke or the heart attack is associated with artery arterioscleroticdiseases, including but not limited to, coronary artery arteriosclerosisand carotid artery stenosis, diabetes, high blood pressure, andperipheral vascular disease.

The invention is defined in its fullest scope in the appended claims andis described below in its preferred embodiments.

DESCRIPTION OF THE FIGURES

The above-mentioned and other features of this invention and the mannerof obtaining them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. These drawings depict only atypical embodiment of the invention and do not therefore limit itsscope. They serve to add specificity and detail, in which:

FIG. 1 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on serum paraoxonase.

FIG. 2 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on their blood pressure.

FIG. 3 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on copper ion-induced LDL oxidation.

FIG. 4 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on LDL basal oxidative state.

FIG. 5A shows the results of carotid intima-media thickness (IMT) inpatients who did not take pomegranate juice. FIG. 5B shows the resultsof carotid IMT after pomegranate juice consumption. FIGS. C and D showsthe results of IMT in the left and right artery respectively afterpomegranate juice consumption..

FIG. 6 shows the results of carotid end diastolic volume (EDV) afterpomegranate juice consumption.

FIG. 7 shows the results of carotid peak systolic volume (PSV) afterpomegranate juice consumption.

FIG. 8 shows the effect of pomegranate juice (PJ) on the carotid lesionplaque composition of patients. FIG. 8A shows the effect of PJconsumption on the cholesterol level of the plaque. FIG. 8B shows theeffect of PJ consumption on lipid peroxides of the plaque. FIG. 8C showsthe effect of PJ consumption on carotid GSH of the plaque. FIG. 8D showsthe effect of PJ consumption on LDL oxidation of the lesion.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a method for treating apatient with arteriosclerosis. The method comprises the step ofadministering to the patient a composition comprising a therapeuticallyeffective amount of an extract from pomegranate.

For the purpose of the present invention, an extract from pomegranatemay be an extract from the whole pomegranate fruit or from anyconstituents of pomegranate fruit. Examples of constituents ofpomegranate fruit that may be used to make the extract of the presentinvention include, but are not limited to, juice, seed, and the innerand outer peel of pomegranate fruit. In one embodiment of the presentinvention, the extract is the juice extract of whole pomegranate fruit.In another embodiment of the present invention, the extract is from theinner or outer peel of pomegranate fruit. In a further embodiment of thepresent invention, the extract may be a mixture of two or more extractsof the whole pomegranate or any constituents of pomegranate.

Methods of making the juice extract of whole pomegranate fruits arecommonly known in the art, and need not be repeated here. In general,any methods that may produce pomegranate juice that naturally occurs inpomegranate may be used. For the purpose of the present invention, thejuice may be concentrated or diluted from its natural concentration. Thejuice may also be mixed with extracts of other constituents ofpomegranate.

Extracts from the constituents of pomegranate, i.e., seeds or the inneror outer peel, may be made by methods commonly known in the art. Forexample, the seeds or the inner or outer peel of pomegranate may bediluted in water and the extract may be made by crushing, squeezing, orextensive vortexing. The insoluble materials of the extract may beseparated from the soluble supernatant of the extract. Preferably, thesupernatant of the extract is used for the purpose of the presentinvention, although any oily, lipidic fraction of the extract may alsobe used. The extract from constituents of pomegranate may beconcentrated or diluted, or mixed with each other or with pomegranatejuice extract.

In accordance with one embodiment of the present invention, the extractof the present invention may be prepared by a process including thesteps of: (a) crushing and squeezing the whole fruits of thepomegranate, including the inner and outer peels and the seeds, to yielda juice component and an insoluble by-product component, and (b)separating the juice component from the insoluble by-product component.The juice component may be used as a juice extract of the presentinvention. The insoluble by-product component may be resuspended in anaqueous medium, such as, but not limited to, water or alcohol, and befurther crushed, squeezed, and mixed to yield a soluble portion and aninsoluble portion. Then the soluble portion may be separated from theinsoluble portion to produce the extract of the constituents of thepresent invention. Alternatively, the soluble portion may be combinedwith the juice extract to produce the extract of the present invention.

In one embodiment of the present invention, the whole fruit of thepomegranate may be enzymatically treated to improve extraction andfiltration. For example, pectinase may be used to treat the whole fruitto prevent the formation of pectin gels. Other enzymes known in the artmay also be used as long as they can improve extraction and filtrationof the extract of the present invention.

The extract of pomegranate of the present invention may be in a liquidor solid form. In accordance with one embodiment of the presentinvention, a solid form of the extract may be made by lyophilizing theliquid extract of the present invention. Alternatively, the constituentsof the pomegranate, such as seeds, inner or outer peels, or anyinsoluble portion discussed above, may be processed directly to form thesolid form of the extract of the present invention. For example, theconstituents of the pomegranate may be dried, and processed into powderor pill forms to be used directly as the solid form of the extract ofthe present invention.

Compositions of the present invention may be a variety of kinds,including, but not limited to, nutritional supplements, pharmaceuticalpreparations, vitamin supplements, food additives, or foods supplements.Compositions of the present invention may be in convenient dosage forms,including, but not limited to, tablets, suspensions, implants,solutions, emulsions, capsules, powders, syrups, liquid compositions,ointments, lotions, creams, pastes, gels, or the like.

Compositions of the present invention may include a carrier. Dependingon the kind of compositions of the present invention, a carrier may be adietary suitable carrier or a pharmaceutically acceptable carrier, aslong as it is compatible with the particular kind of compositions of thepresent invention. Examples of a dietary suitable carrier include, butare not limited to, dietary suitable excipients, diluents, and carriers.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. As used herein, theterms “pharmaceutically acceptable,” “physiologically tolerable,” andgrammatical variations thereof, as they refer to compositions, carriers,diluents, and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a mammal without theproduction of undesirable physiological effects.

The compositions of the present invention may be used alone or incombination with other biologically active ingredients. A composition ofthe present invention, alone or in combination with other activeingredients, may be administered to a subject in a single dose ormultiple doses over a period of time, generally by oral administration.Various administration patterns will be apparent to those skilled in theart. The dosage ranges for the administration of the compositions of thepresent invention are those large enough to produce the desired effect.The dosage should not be so large as to cause any adverse side effects,such as unwanted cross-reactions and the like. Generally, the dosagewill vary with the age, weight, sex, condition, and extent of acondition in a subject, and the intended purpose. The dosage can bedetermined by one of skill in the art without undue experimentation. Thedosage can be adjusted in the event of any counter indications,tolerance, or similar conditions. Those of skill in the art can readilyevaluate such factors and, based on this information, determine theparticular effective concentration of a composition of the presentinvention to be used for an intended purpose.

In one embodiment of the present invention, a composition contains theextract of pomegranate in a dosage unit in an amount that contains atleast 30 to 3000 μmols per dosage unit of polyphenols. For the purposeof the present invention, polyphenols are those naturally present in theextract of pomegranate. It should be appreciated that polyphenols areused herein as a measurement marker for the amount of extract that needsto be used in each dosage unit. They are not used herein as anindication that they are the active, or the only active, ingredients ofthe extract. In fact, it is possible that something else, or the synergyof polyphenols and other components of an extract of the presentinvention, may be responsible for the activities of the extract.

The term “dosage unit” as used herein refers to physically discreteunits suitable as unitary dosages for animals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent,e.g., a carrier or vehicle. The specifications for the unit dose of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and (b) the limitations inherentin the art of compounding such active material for therapeutical use inanimals.

The term “therapeutically effective amount” as used herein means thatthe amount of the extract of the present invention contained in thecomposition administered is of sufficient quantity to achieve theintended purpose, such as, in this case, to cause regression ofarteriosclerosis in the patient. For the purpose of the presentinvention, regression of arteriosclerosis may be measured by thedecrease in the size of lesions caused by arteriosclerosis. Methods ofmeasuring the lesion size is well known in the art, and need not berepeated herein. For example, ultrasound and angiography methods may beused to measure the lesion size. In accordance with one embodiment ofthe present invention, the regression is determined by measuring theintima-media thickness of an artery, such as, but not limited to,carotid artery or coronary artery.

Accordingly, by determining the regression of arteriosclerosis in apatient, one skilled in the art can readily determine whether the amountof the extract of the present invention is therapeutically effective inview of the disclosure of the present invention without undueexperimentation. In one embodiment, the therapeutically effective amountof the extract of the present invention contains at least 30 to 3000μmols of polyphenols naturally occurring in a pomegranate fruit. Again,it should be appreciated that the polyphenols are used herein as ameasurement marker for the concentration of the extract of the presentinvention. In another embodiment, the composition contains one glass ofjuice extract of the present invention.

The method of the present invention may be used to treat patients witharteriosclerosis, particularly patients with carotid or coronary arteryarteriosclerotic diseases. In addition, the methods of the presentinvention may be used to treat patients with an increased intima-mediathickness of an artery, particularly to treat patients with an increasedintima-media thickness of carotid artery or coronary artery caused bycarotid artery arteriosclerosis or coronary artery arteriosclerosis,respectively. Furthermore, since artery arteriosclerosis is closelyassociated with the incidence of stroke and heart attack, the methods ofthe present invention may also be used to decrease the incidence ofstroke and heart attack in a patient, particularly those associated withcarotid or coronary artery arteriosclerotic disease.

Accordingly, another aspect of the present invention provides a methodof treating a patient with an increased intima-media thickness of anartery. The method comprises the step of administering to the patient acomposition comprising an amount of an extract from pomegranate which istherapeutically effective to reduce the intima-media thickness of theartery. The term “therapeutically effective” as used herein means thatthe amount of the extract of the present invention contained in thecomposition administered is of sufficient quantity to reduce theintima-media thickness of the artery. According to one embodiment of thepresent invention, the intima-media thickness of the artery may be dueto arteriosclerosis, such as, but not limited to, coronary arteryarteriosclerosis and carotid artery stenosis, diabetes, high bloodpressure, and peripheral vascular disease.

A further aspect of the present invention provides a method ofdecreasing the incidence of stroke or heart attack in a patient. Themethod comprises the step of administering to the patient a compositioncomprising a therapeutically effective amount of an extract frompomegranate to decrease the incidence of stroke or heart attack in apatient. As used herein, the term “therapeutically effective” means thatthe amount of the extract of the present invention contained in thecomposition administered is of sufficient quantity to decrease theincidence of stroke or heart attack in a patient. The decrease in thesize of the plaque or increase in the stability of the plaque at thelesion of a patient may decrease the incidence of stroke or heartattack. According to one embodiment of the present invention, the strokeor the heart attack may be associated with arteriosclerotic diseases,including, but not limited to, carotid or coronary arteryarteriosclerotic disease, and peripheral vascular disease.

The following examples are intended to illustrate, but not to limit, thescope of the invention. Indeed, those of ordinary skill in the art canreadily envision and produce further embodiments, based on the teachingsherein, without undue experimentation.

EXAMPLES Methods

Pomegranate Processing

Pomegranates were picked by hand, washed, chilled to 32° F., and storedin tanks. Then the fruit was crushed, squeezed, and enzymaticallytreated with pectinase to yield the pomegranate juice and theby-products, which include the inner and outer peels and the seeds.Pectinase hydrolyzes alpha-1.4 galacturonide bonds in pectin and, thus,it improves extraction and filtration, and prevents formation of pectingels. The juice was filtered, pasteurized, concentrated, and stored at−18° C.

Alternatively, pomegranates were picked by hand, washed, and stored intanks. Then, the fruits were crushed and squeezed. The juice wasfiltered, pasteurized, concentrated, and stored at −18° C. Each dayduring the study period, the concentrated PJ was diluted 1:5 (v:v) withwater in order to obtain a single strength PJ.

Peels and Seeds Extracts

One gram of inner or outer peels/seeds was diluted in 5 ml of waterfollowed by crushing, squeezing, and extensive vortexing. Then, theextract was centrifuged to remove any water insoluble materials and thesupernatant was used alone or mixed with the juice extract for use inthe methods of the present invention.

In the seeds extraction, an upper oily, lipidic fraction appeared whichwas not used in the study of aqueous extracts, but may also contain someactive compounds. Therefore, the oily, lipidic fraction may also be usedas an extract of the present invention.

Polyphenols Determination

Total polyphenols concentration in pomegranate juice was determinedspectrophotometrically with the phosphomolybdic phosphotungstic acidreagents (26).

LDL Isolation

LDL is isolated from blood samples by discontinuous density gradientultracentrifugation and is then dialyzed against saline with EDTA (1mmol/L) (14). Before the oxidation study, LDL is diluted in phosphatebuffered saline (PBS) to 100 mg protein/L and dialyzed overnight againstPBS at 4° C. to remove the EDTA. LDL protein concentration is determinedwith the Folin phenol reagent (15).

LDL Oxidation

Oxidation of LDL is carried out in a shaking water bath at 37° C. underair, in plastic tubes, 1 cm in diameter. For metal ion-dependentoxidation, LDL is incubated for 4 hours at 37° C. with freshly preparedCuSO₄ (5 μmol/L).

B-Mode Ultrasonography

Common carotid artery IMT from B-mode ultrasound is a widely usedmeasure of early atherosclerosis (36-38). After a careful axial scan,longitudinal B-mode images of common carotid artery wall boundaries wereobtained with a high resolution (7 MH_(z) linear transducer, Acuson128xP, Acuson Co, Mountain View, Calif.). Maximum IMT was measured atthe far wall of the distal common carotid arteries, about one centimeterfrom the carotid bifurcation. Atherosclerotic plaques at the commoncarotid arteries and the carotid bulb, as well as the proximal anddistal internal carotid arteries were imaged and the length and width ofthe plaque were assessed. On duplex examination of the internal carotidarteries, flow velocities were calculated at the stenotic sites, andexpressed by peak systolic velocity (PSV) and EDV. The ultrasoundoutcome analyses were the change over time in the maximal IMT among thesame preselected carotid artery segment, and the change in the plaquedimensions and in blood flow velocities. Patients had up to five B-modeand duplex examinations of the common and internal carotid arteries onboth sides. There was one ultrasound examination at baseline, one every3 months and one at the final clinical visit after 12 months of PJconsumption. All ultrasound studies were done by the same physicianassuring reproducibility of the site of IMT and plaques measurements, aswell as site and interrogation angles on the duplex follow-upexaminations. The sonologist was aware of the results of measurements onprevious examinations. In order to avoid the potential introduction ofscanner-dependent variabilities, the same ultrasound system was used forindividual patients on follow-up examinations.

For metal ions-dependent oxidation, LDL (100 μg of protein/ml) wasincubated for 2 hours at 37° C. with freshly prepared CuSO₄ (5 μM). LDLoxidation was terminated by refrigeration at 4° C., and the addition of0.1 mM Na₂ EDTA and 25μM BHT to the CuSO₄ system. The formation ofconjugated dienes was continuously monitored by measuring the increasein absorbency at 234 nm (60). The lag time required for initiation oflipoprotein oxidation was calculated from the oxidation curve. Theamount of LDL-associated lipid peroxides was measured by the method ofEI-Saadani et al. (39).

For metal ions-independent oxidation, LDL is incubated for 2 hours at37° C. with 5 mM AAPH. Na₂ EDTA (0.1 mM) is added to the incubationmedium in order to chelate adventitious metal ions that could otherwisecontribute to the radical initiator-induced oxidation. LDL oxidation isterminated by refrigeration at 4° C. LDL oxidation is determined bymeasuring the amount of thiobarbituric acid reactive substances (TBARS),lipid peroxides formation, and by continuous monitoring of the formationof conjugated dienes by measuring the increase in absorbance at 234 nm.

For macrophage-mediated oxidation, J-774 A.1 macrophages (2×10⁶ cells/35mm dish, obtained from the American Tissue Culture Collection,Rockville, Md.), is incubated with LDL (100 μg of protein/ml) for 18hours at 37° C. in medium Ham's F-10, in the presence of 2 μM CuSO₄. Theextent of LDL oxidation is measured directly in the medium by the TBARSassay. LDL incubated under similar conditions in the absence of cells(cell-free). serves as a control. Cell-mediated oxidation of LDL iscalculated by subtracting the cell-free value from the value obtained inthe presence of cells. LDL oxidation is determined by measuring theamount of thiobarbituric acid reactive substances (TBARS) (16).

The formation of conjugated dienes is continuously monitored bymeasuring the increase in absorbance at 234 nm (17). Incubations arecarried out in the spectrophotometer cuvette at 37° C. in a thermostaticsix-cell holder in a spectrophotometer (Ultrospec 3000, Pharmacia,Biotech). The reference cell contains 5 μmol/L CuSO₄ in PBS, or 1 mmol/Lof AAPH, together with 0.1 mM EDTA in PBS, for metal ion dependent ormetal ion-independent oxidation, respectively. The increase inabsorbance during LDL oxidation is recorded over time every ten minutes.

The LDL-associated lipid peroxide formation is determined with acommercially available kit (cholesterol color reagent, CHOD iodidemethod, Diagnostica Merck, Darmstadt, Germany) (18). This assay is basedon the oxidative activity of lipid peroxides that convert iodide toiodine and is measured spectrophotometrically. The assay is performedwith 100 μl of lipoprotein solution (100 mg protein/ml) which is mixedon a vortex with 1 ml color reagent. The sample is allowed to stand for30 minutes in the dark, followed by a determination at 365 nm against ablank solution.

LDL Aggregation

LDL (100 μg of protein/mL) is mixed by vortex at a fixed strength, andthe absorbance at 680 nm is monitored every 10 seconds against a blanksolution (19). LDL aggregation is also induced by its subjection to theaction of sphyngomyelinase (SMase) or phospholipase C (PLase C) (61).

LDL Retention

LDL (200 μg of lipoprotein protein/mL) is incubated with chondroitinsulfate (CS, 100 μg/mL) for 30 minutes at room temperature. Thelipoprotein is precipitated with a commercial kit for HDL cholesterolreagent (phosphotungstic acid/MgCl₂, Sigma Co., St. Louis, Mo.) thatprecipitates all the LDL present in the samples, followed by a 10-minutecentrifugation at 2000×g (20). After discarding the supernatant, the LDLin the precipitate is dissolved in 0.1 N NaOH, and analyzed for itsglycosaminoglycans (GAGs) content using the 1,9-dimethylmethylene blue(DMMB) spectrophotometric assay for sulfated glycosaminoglycans (21).The absorbency at 525 nm is then immediately measured.

Paraoxonase Activity

Paraoxonase is determined as its arylesterase activity (22). Arylestraseactivity is measured using phenylacetate as the substrate. The initialrates of hydrolysis are determined spectrophotometrically at 270 nm. Theassay mixture includes 1.0 mM phenylactate and 0.9 mM CaCl₂ in 20 mMTris HCl, pH 8.0. Non-enzymatic hydrolysis of phenylacetate issubtracted from the total rate of hydrolysis. The E₂₇₀ for the reactionis 1,310 M⁻¹ cm⁻¹. One unit of arylesterase activity is equal to 1 μmolof phenylacetate hydrolyzed/min/ml. A purified enzyme has nearly 2000units of arylesterase activity per mg protein.

Human Studies

Nineteen patients, 5 females and 14 males, ages 65-75 years, withasymptomatic severe carotid artery stenosis (CAS, defined as 71-90%stenosis of one of the carotid arteries), were included in the study.These patients had an abnormal echo doppler of the carotids, which wasperformed following a finding of carotid murmur in physiologicalexamination, or complaints of headache or dizziness. Their dietaryhabits and life style did not change during the study. The patients wereunder several hypotensive drugs, and continued this therapy along thestudy. Nine patients that did not consume PJ served as the controlgroup. In these patients, the echo doppler of the carotid arteries wasperformed at the beginning of the study and after one year. Ten patientsconsumed 1 glass of PJ (1:5, v:v dilution of concentrated PJ whichcontained 1.5 mmoles total polyphenols equivalents) in the evening for aperiod of up to 12 months. Blood analyses and echo doppler of thecarotid arteries were performed at the beginning of the study and at 3,6, 9, and 12 months after starting PJ consumption. Blood samples werealso taken 1 month after the termination of PJ consumption. Bloodpressure was monitored in the morning in a sitting position. The studywas approved by the Helsinki Committee of the Rambam Medical Center,Israel Ministry of Health.

Echo Doppler Evaluation

All patients had a complete echo doppler evaluation of the carotidarteries including intimal-medial thickness. The ultrasonographic imagesof both carotid arteries were analyzed before pomegranate juiceconsumption, and after 3, 6, 9, and 12 months of juice consumption. Theexaminations were performed by B-mode high resolution ultrasonographywith a 7-10 MHz transducer. The intima-media complex thickness wasmeasured at the common carotid artery 2 cm proximal to the bifurcation(23, 24). Each atherosclerotic plaque, both in the common and in theinternal carotid artery, was imaged for size and configuration of theplaque (soft, calcified, ulcerated). Progression/regression ofstabilization of carotid arteriosclerosis was determined for each plaqueand intima-media thickness measurement. A pulsed echo dopplerexamination combined with a B-mode ultrasound (Duplex) was performed atstenotic artery segments. Peak systolic and diastolic velocities weremeasured and internal carotid/common carotid ratios were thencalculated.

Blood Analysis

1. Biochemical tests in serum, which includes the measurement of serumglucose, createnine, urea, billirubin, creatine kinase, and aspartateamino transferase by conventional means known in the art.

2. Liver function tests by known methods.

3. Kidney function tests by known methods.

4. Plasma lipids profile, including total cholesterol, LDL-, VLDL-, andHDL-cholesterol and total triglycerides phospholipids, apolipoproteinsA-I and apolipoprotein B-100 concentrations. Known conventional meansare used to conduct the measurements.

5. Plasma susceptibility to oxidation induced by a free radicalgenerating system, AAPH (2,2′-azobis 2-amidinopropane hydrochloride),which is an aqueous soluble azo compound that thermally decomposes toproduce peroxyl radicals at a constant rate. Plasma susceptibility tooxidation is analyzed following incubation of 1 ml of plasma for 2 hoursat 37° C. with 100 mM AAPH. The formation of thiobarbituric acidreactive substances (TBARS) and of lipid peroxides is measured incomparison to plasma that is incubated under similar conditions, butwithout AAPH.

6. Total antioxidant status (TAS) in plasma is evaluated by methodsknown in the art. Total antioxidant status was measured in serum with acommercially available kit (catalog no. NX 2332; Randox Laboratories,Antrim, United Kingdom, 19).

7. Serum anti Ox-LDL antibodies: The amount of serum anti Ox-LDLantibodies was measured in serum samples collected from the patientsbefore treatment and after 3 or 6 months of PJ consumption, by using theimmunoelisa anti-Ox-LDL test (Immco Diagnostics, Inc. Buffalo, N.Y.,USA, Cat. No 1158). Results are expressed as Enzyme Units per milliliter(EU/ml).

8. Total polyphenols in plasma is measured as described above.

9. Paraoxonase (an HDL-associated esterase that protects from lipidperoxidation) activity in serum. Paraoxonase is determined as itsarylesterase activity described above.

10. Separation of VLDL, LDL and of HDL from plasma byultracentrifugation and determination of their lipid content.

Plasma samples drawn from the patients before and after 1, 3, 6, 9, and12 months of PJ consumption were stored at −20° C. until all sampleswere collected. Plasma lipoproteins (LDL, VLDL, HDL) were isolated bysequential density ultra-centrifugation (19). The LDL was washed atd=1.063 g/mL, and dialyzed against 150 mmol/L NaCI and 1 mmol/L Na₂ EDTA(pH 7.4) at 4° C. The LDL fractions were then sterilized by filtration(0.45 μm), kept under nitrogen in the dark at 4° C., and used within 2weeks. The lipoprotein protein concentration was determined by the Lowryassay (59). Prior to oxidation, LDL was dialyzed against EDTA-free,phosphate-buffered saline (PBS) solution at pH 7.4, and at 4° C. Alllipoproteins were analyzed for their content of protein, total andunesterified cholesterol, triglycerides, phospholipids, vitamin E, andcarotenoids.

11. Basal Oxidative State of LDL including measurement of cholesterylester and cholesteryl ester hydroperoxides by HPLC, as discussed above.

12. LDL Susceptibility to Oxidation. LDL is dialyzed for 24 hoursagainst phosphate buffered saline (PBS) before oxidation to remove theEDTA. Oxidation of LDL is carried out in a shaking water bath at 37° C.under air. LDL is subjected to oxidation by copper ions, by AAPH, aswell as by macrophages in cell culture.

13. Serum Angiotensin II Converting Enzyme (ACE) Activity. ACE activityis analyzed spectrophotometrically in order to evaluate possible effectsof pomegranate juice on atherosclerosis via this pathway.

14. Carotid Lesion: Complete atherosclerotic plaques, including thecommon internal and external carotid parts of the plaque, were removedfrom seven patients that did not consume PJ and from two patients thatconsumed PJ for 3 or 12 months. Lesions were washed in saline, dried,and their weight measured. The lesions were cut into small pieces andrinsed in PBS, followed by their sonication in an ultrasonic processor(3×20 seconds at 80 W). The lesion's cholesterol content was measured inthe homogenate samples by an enzymatic, colormetric assay using acommercial kit (Sigma Co. Ltd). Lesion's lipid peroxides content wasalso measured (41). Reduced glutathione (GSH) content was measured bythe (DTNB)-GSSG reductase recycling assay (42).

15. Statistics: The ANOVA test was performed for all statisticalanalyses. Results are given as the mean±SEM. Assays in each sample wereperformed in triplicate. All comparisons are shown for data after PJconsumption vs. the results obtained before treatment.

Results

The results of the echo doppler evaluation of the seven male and threefemale patients with asymptomatic severe carotid stenosis are summarizedin Table 1. Table 1 shows the effect of pomegranate juice consumption onintima-media thickness (IMT) of the patients with carotid arterystenosis. The results indicate that pomegranate juice consumption by thepatients can reduce the size of lesions already formed. The intima-mediathickness of the carotid artery showed regression of the size of thelesions.

TABLE 1 The Effect of Pomegranate Juice Consumption on Intima-MediaThickness (IMT) in Patients with Carotid Artery Stenosis Patient (#) 0 3Months 6 Months 9 Months 12 Months A. Right 1 1.7 1.3 1.2 1.1 0.8 2 1.31.3 1.2 1.0 0.8 3 1.3 1.3 1.2 0.7 0.6 4 2.7 2.7 2.2 2.0 1.4 5 2.8 2.82.7 2.1 1.2 6 1.8 1.8 1.3 1.3 0.9 7 1.9 1.9 1.7 1.1 0.9 8 3.8 3.8 2.21.9 1.7 9 1.8 1.7 1.5 1.0 0.8 10 1.9 1.9 1.6 1.5 1.2 B. Left 1 1.1 1.11.0 0.8 0.6 2 1.0 1.0 0.9 0.8 0.6 3 1.3 1.2 0.9 0.7 0.6 4 2.3 2.3 2.01.4 1.1 5 2.3 2.3 1.7 1.2 0.7 6 2.3 2.2 1.3 1.1 0.8 7 2.5 2.2 2.1 1.91.2 8 3.7 2.4 1.9 1.6 1.0 9 2.8 2.6 2.4 2.3 1.8 10 2.8 2.6 2.5 2.3 1.8

The effect of pomegranate juice (PJ) consumption by patients withcarotid artery stenosis the lesion content of lipids, oxidized lipids,GSH and on the lesion-mediated oxidation of LDL is also measured, andthe results are summarized in Table 2. Lesions were obtained frompatients with carotid artery stenosis; seven of the patients did notconsume PJ (control), and two of the patients consumed PJ for 3 or 12months. The amount of cholesterol, triglyceride, lipid peroxides, andGSH (reduced glutathione) were measured in the lesions homogenates asdescribed above. Lesions (0.3 g) were incubated with LDL

TABLE 2 The effect of pomegrante juice (PJ) consumption by patients withcarotid artery stenosis on lesion content of lipids, oxidized lipids,GSH and on lesion-mediated oxidation of LDL. Lipid Lesion- Cho- Trigly-Peroxides GSH Mediated Oxi- esterol ceride (nmol/ (nmol/ dation of(mg/mg (mg/mg mg mg LDL(nmol lesion lesion lesion lesion peroxides/mgprotein) protein) protein) protein) LDL protein) Control 0.59 ± 0.33 ±219 ± 0.26 ± 318 ± 24 Lesions 0.14 0.07 17  0.04 (n = 7) Lesion after 30.23 ± 0.20 ±  85 ± 0.63 ± 181 ± 11 months of PJ 0.06 0.03 5 0.05consumption (n = 1) Lesion after 12 0.45 ± 0.09 ± 123 ± 0.67 ± 216 ± 15months of PJ 0.08 0.01 8 0.02 consumption (n = 1)

(100 μg of protein per ml) in PBS for 20 hours at 37° C. LDL was alsoincubated under the same conditions without lesions. The extent of LDLoxidation was measured by the lipid peroxide assay. Lesion-mediatedoxidation of LDL was calculated by subtracting the values obtained incontrol LDL from those obtained upon incubation of LDL with the lesions.Results summarized in Table 2 indicate that after pomegranate juiceconsumption, there was a decrease in total cholesterol, triglycerides,and lipid peroxides. LDL oxidation was also diminished. However, therewas an increase in GSH. The analysis indicates that there werebeneficial quantitative and qualitative changes in carotid arterylesions. In order to analyze the effect of PJ consumption on thepatients' serum oxidative state, we measured the serum concentration ofantibodies against. Ox-LDL. A significant (p<0.01) reduction in theconcentration of antibodies against Ox-LDL by 24% and 19% was observedafter 1 and 3 months of PJ consumption, respectively, (from 2070±61EU/ml before treatment to 1563±69 and 1670±52 EU/ml after 1 and 3 monthsof PJ consumption, respectively). Total antioxidant status (TAS) inserum was substantially (2.3 fold) increased from 0.95±0.12 nmol/L atbaseline, up to 2.20×0.25 nmol/L after 12 months of PJ consumption.These results indicate that PJ administration to the patientssubstantially reduced their serum oxidative stress, and could thusinhibit plasma lipid peroxidation. The susceptibility of the patients'plasma to AAPH-induced oxidation was reduced after 12 months of PJconsumption by 62% (from 209±18 at baseline to 79±6 nmol of peroxides 1ml after 12 months of PJ consumption. We next isolated the patients' LDLand analyzed their basal oxidative state, as well as the susceptibilityof their LDL to copper ion-induced oxidation (FIGS. 1, 3 and 4).

FIG. 1 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on serum paraoxonase. FIG. 1 demonstrates asignificant (p<0.01) increase in serum paraoxonase, measured asarylesterase activity, by 11%, 42%, 49%, and 83% after 3, 6, 9 and 12months of PJ consumption, respectively. The results indicate thatpomegranate juice consumption can significantly increase serumarylesterase activity (a major activity of the enzyme paraoxonase).

FIG. 2 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on their blood pressure. After 12 months ofPJ consumption, the patients' systolic blood pressures weresignificantly reduced by 18% compared to the values obtained beforetreatment (FIG. 2A). The results show that pomegranate juice consumptionhas no significant effect on diastolic blood pressure (FIG. 2B).

FIG. 3 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on copper ion-induced LDL oxidation. Thesusceptibility of LDL to copper ion-induced oxidation was graduallyreduced, as observed by prolongation of the lag time required for theinitiation of LDL oxidation (from 30±4 minutes before treatment to 55±5,60±2, 64±1, and 65±2 minutes after 3, 6, 9, and 12 months of PJconsumption, respectively) (FIG. 3A). The LDL lipid peroxides contentformed during copper ion-induced LDL oxidation was also reduced by 40%,49%, 57%, and 59% after 3, 6, 9, and 12 months of PJ consumption,respectively (FIG. 3B). The results show that pomegranate juiceconsumption has an inhibitory effect on LDL oxidation.

FIG. 4 shows the effect of pomegranate juice consumption by patientswith carotid artery stenosis on the LDL basal oxidative state. PJconsumption resulted in a significant reduction in the levels ofLDL-associated lipid peroxides by 43%, 89%, 86%, and 90% after 3, 6, 9,and 12 months of PJ consumption, respectively. The results show thatpomegranate juice consumption has inhibitory effect on the LDL basaloxidative state.

FIG. 5 shows the results of carotid IMT after pomegranate juiceconsumption. Intima-media thickness (IMT) in the more pathologicalcarotid artery (left or right) from the control group increasedsignificantly (p<0.01) during one year by 10% (FIG. 5A), whereas inpatients that consumed PJ, a significant (p<0.01) and substantialreduction in the IMT, by 43%, was noted after one year of PJ consumption(FIG. 5B). Analysis of carotid IMT before and during PJ consumptionrevealed a gradual reduction of 20%, 27%, 28%, and 38% in the leftcarotid artery (FIG. 5C), and a reduction of 6%, 25%, 28%, and 32% inthe right carotid IMT (FIG. 5D), which was observed after 3, 6, 9, and12 months of PJ consumption, respectively, in comparison to “0 time”.

FIG. 6 shows the results of carotid end diastolic volume (EDV) afterpomegranate juice consumption. The results show that pomegranate juiceconsumption can reduce the velocityin the left carotid artery by 11%,14%, 17%, and by 33% (FIG. 6A), and in the right carotid artery by 20%,26%, 40%, and 52% (FIG. 6B) after 12 months of pomegranate juiceconsumption. This confirms the fact that the internal diameter of thecarotid artery increases after pomegranate juice consumption.

FIG. 7 shows the results of carotid peak systolic volume (PSV) afterpomegranate juice consumption. The results show that pomegranate juiceconsumption can reduce the velocity by 12% and 28% in the left (FIG. 7A)and the right (FIG. 7B) carotid arteries, respectively, after 12 monthsof pomegranate juice consumption. Again, this confirms the fact that theinternal diameter of the carotid artery increases after pomegranatejuice consumption.

FIG. 8 shows the effect of pomegranate juice (PJ) consumption bypatients with carotid artery stenosis on lesion content of lipids,oxidized lipids, GSH, and on lesion-mediated oxidation of LDL. In thegroup of ten patients with carotid lesions who consumed PJ, one of themhad to undergo carotid lesion removal at three months. Another one hadto have carotid lesion removed at 12 months. The remaining patients didnot need any operations. However, among the 9 patients who did not takejuice extract of pomegranate, seven of the 9 patients needed to haveoperations to remove their carotid lesions. After the carotid lesionswere obtained from the above patients, the level of cholesterol, carotidlipid peroxides, carotid GSH, and carotid lesion-included LDL oxidationin the removed carotid lesions were measured. The results are summarizedin FIG. 8 as FIGS. 8A to D, respectively.

The cholesterol content in the lesions was lower after PJ consumption incomparison to lesions from patients that did not consume PJ (FIG. 8A).The lipid peroxides content in lesions after PJ consumption for 3 or 12months was significantly reduced by 61% or 44%, respectively, ascompared to control lesions from untreated patients (FIG. 8B). As thebalance between lesion pro-oxidants and anti-oxidants levels determinesthe extent of oxidized lipids, which accumulate in the lesion, we nextdetermined the levels of reduced glutathione (GSH, the major cellularantioxidant) (43). A substantial increase in lesion GSH content, by2.5-fold, was observed after PJ consumption (3 or 12 months, FIG. 8C).LDL oxidation by lesions derived from the patients after PJ consumptionfor 3 or 12 months (with no addition of copper ions), measured by theamount of lipid peroxides, was significantly (p<0.01) decreased by 43%or 32%, respectively, in comparison to LDL oxidation by lesions fromuntreated patients (FIG. 8D).

The results show that there is definitely a primary qualitative changein the actual lesions in people who did receive pomegranate juice.Clearly, the extract of the present invention changes the physicalcompositions of the plaque, and the oxidation. and anti-oxidationactivities within the plaque.

DISCUSSION

The present study clearly demonstrates, for the first time, thatpomegranate juice (PJ) consumption by patients with carotid arterystenosis (CAS) resulted in the following changes: a reduction in carotidIMT, a decrement in systolic blood pressure, and an inhibition in LDL.oxidation. Although the use of a small number of patients could cause astatistical error, we performed power calculations using ANOVA tests andrunning each sample in triplicate. All the above results clearlydemonstrate statistically significant anti-atherogenic effects ofpomegranate juice consumption in patients with CAS.

The effect of PJ consumption on carotid lesion progression in thesepatients was assessed by measuring IMT, PSV, and EDV in the right andleft carotid arteries before, during, and after one year of PJconsumption. A marked decrement in IMT and blood flow velocities weredemonstrated after 12 months of PJ consumption, compared to valuesobtained at the baseline. The average IMT varies in middle aged menbetween 0.7 to 1.2 mm, and the progression slope of the mean maximumcarotid IMT in untreated patients with carotid artery stenosis was shownto be about 0.02 mm/year (44).

In the present study, PJ consumption not only prevented the progressionof the lesion, as compared to IMT in the control group, but alsoresulted in a reduction in the lesion size. A reduction in oxidativestress was demonstrated already after 1 month of PJ consumption, but theinhibitory effects of PJ consumption on carotid lesion parameters weredemonstrated only after 9-12 months. This may indicate that if the PJantioxidant properties are responsible for the lesion size regression,then a long period of reduced oxidative stress is required to affectcarotid lesion size. In recent studies, it was demonstrated that reducedlevels of antioxidants are associated with increased IMT (45), and apositive association was observed between antibodies to oxidized LDL andIMT in healthy aged men (46).

The lipid peroxidation hypothesis of atherosclerosis (2, 3, 47) issupported by the presence of oxidized lipids in atherosclerotic lesions,increased oxidative states of LDL from atherosclerotic patients (4, 48),and the anti-atherogenicity of antioxidants against LDL oxidation (4,5). The impressive ability of PJ to inhibit LDL oxidation (basal andcopper ion-induced) could be related to the high potency of PJ tanninsto scavenge-free radicals (12, 13). Copper ion-induced LDL oxidationresulted in similar changes in the lipoprotein to those observed inarterial wall oxidized LDL-Iike particles (49, 50).

Flavonoids, as a more potent antioxidant, may overcome the limitationsof vitamin E and carotenoids, and pomegranate juice as a source of oneof the most potent antioxidants, such as hydrolyzable tannins andanthocyanin (12).

Serum paraoxonase (PON1) activities (arylesterase/paraoxonase) arereduced in atherosclerotic patients, such as hypercholesterolemic,diabetics, and patients with vascular diseases (51, 52). We havepreviously shown increased serum PON1 activity in healthy humanvolunteers upon PJ consumption (13). Similarly in the present study, wealso showed that PJ consumption by patients with CAS resulted in asubstantial increase in serum PON1 activity (up to 83% after one year).PON1, an HDL-associated esterase, protects lipoproteins from oxidation(7, 10). This protection is probably the result of paraoxonase abilityto hydrolyze specific oxidized lipids in oxidized lipoproteins and inhuman atherosclerotic lesions (7-9). Paraoxonase is inactivated byoxidized lipids (53), and red wine flavonoids (53), as well aslicorice-derived isoflavin glabridin (53), can preserve paraoxonaseactivity during lipoprotein oxidation. The increase in serum paraoxonaseactivity may be a direct effect of PJ, as we have previouslydemonstrated (13), or it might also be the result of the reduction inlipid peroxides, by PJ antioxidants. PJ contains very potentantioxidants and, unlike other nutrients (53), it not only preservesserum PON1, but also even increases the enzyme activity (13). Anotheranti-atherogenic effect of PJ consumption is its effect on bloodpressure, as the patients' systolic blood pressure was significantlyreduced after 12 months of PJ consumption. Similar results were recentlyobtained upon PJ administration to hypertensive patients (54). Asreactive oxygen species contribute to endothelium-dependent contraction,antioxidants can possibly restore endothelial function, and hence, maydecrease blood pressure (55, 56). The decrement in the systolic bloodpressure upon PJ consumption may also be associated with reduced IMT(57).

Finally, the findings of reduced oxidative stress in lesions obtainedfrom patients that consumed PJ, in comparison with lesions fromuntreated patients, could possibly be related to the effect ofPJ-induced increased paraoxonase hydrolytic activity on the lesions'oxidized lipids (7-9). This phenomenon shows that, in addition to theregression of carotid lesion size, the lesion itself may be consideredless atherogenic after PJ consumption, as its lipids and oxidized lipidscontent decreased, and since its ability to oxidize LDL wassignificantly reduced. Morphologically, the lesions obtained from thepatients that consumed PJ were visually less fatty and fragile (data notshown), and may represent macrophage foam cells-poor carotid plaques.

It is thus concluded that, as previously shown in atherosclerotic mice(13, 58), pomegranate juice consumption by patients with carotid arterystenosis also possesses an impressive anti-atherosclerotic properties,as it substantially decreases serum oxidative stress and reduces carotidintima-media thickness.

It is an unexpected discovery of the present invention that pomegranatejuice consumption can cause regression in the lesion size of carotidartery arteriosclerosis. Prior research has shown a parallel betweencarotid artery arteriosclerotic disease and coronary arteryarteriosclerotic disease (27 to 35). Therefore, it is believed thatpomegranate juice consumption would also lead to regression in coronaryartery disease. In addition, due to the known relationship of thecarotid and coronary artery arteriosclerotic diseases with the strokeand heart attack, it is believed that the pomegranate extract of thepresent invention may also be used to reduce the incidence of stroke andheart attack in a patient.

The present invention may be embodied in other specific forms withoutdeparting from its essential characteristics. The described embodimentis to be considered in all respects only as illustrative and not asrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changeswhich come within the meaning and range of the equivalence of the claimsare to be embraced within their scope.

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What is claimed is:
 1. A method of treating a patient witharteriosclerosis, comprising the step of administering to the patient acomposition comprising a therapeutically effective amount of an extractfrom pomegranate, wherein the extract is prepared by a processcomprising the steps of: (a) crushing and squeezing the whole fruits ofpomegranate including inner and outer peels and the seeds to yield ajuice component and an insoluble by-product component; and (b)separating the soluble juice component from the insoluble by-productcomponent and removing the insoluble by-product component therefrom toproduce the extract.
 2. The method of claim 1, wherein the extract is ajuice extract of pomegranate fruit.
 3. The method of claim 1, whereinthe extract is from inner or outer peel of pomegranate fruit.
 4. Themethod of claim 1, wherein the extract contains at least 30 to 3000 μmolof polyphenols naturally present in the extract of pomegranate.
 5. Themethod of claim 1, wherein the composition further comprises apharmaceutically acceptable carrier.
 6. The method of claim 1, whereinthe composition is administered orally.
 7. The method of claim 1,wherein the composition is administered in a form selected from a groupconsisting of tablets, suspensions, implants, solutions, emulsions,capsules, powders, syrups, liquid compositions, ointments, lotions,creams, pastes, gels, and the like.
 8. The method of claim 1, whereinthe patient has carotid artery arteriosclerotic disease.
 9. The methodof claim 1, wherein the patient has coronary artery arterioscleroticdisease.
 10. A method of treating a patient with an increasedintima-media thickness of an artery, comprising the step ofadministering to the patient a composition comprising an amount of anextract from pomegranate which is therapeutically effective to reducethe intima-media thickness of the artery, wherein the extract isprepared by a process comprising the steps of: (a) crushing andsqueezing the whole fruits of pomegranate including inner and outerpeels and the seeds to yield a juice component and an insolubleby-product component; and (b) separating the soluble juice componentfrom the insoluble by-product component and removing the insolubleby-product component therefrom to produce the extract.
 11. The method ofclaim 10, wherein the patient has arteriosclerotic disease.
 12. Themethod of claim 10, wherein the patient has coronary arteryarteriosclerotic disease.
 13. The method of claim 10, wherein thepatient has carotid artery stenosis.
 14. The method of claim 10, whereinthe extract is a juice extract of pomegranate fruit.
 15. The method ofclaim 10, wherein the extract is from inner or outer peel of pomegranatefruit.
 16. The method of claim 10, wherein the extract contains at least30 to 3000 μmol of polyphenols naturally occurred in the extract ofpomegrantate.
 17. The method of claim 10, wherein the composition isadministered orally.
 18. A method of decreasing the incidence of strokeor heart attack in a patient, comprising the step of administering tothe patient a composition comprising a therapeutically effective amountof an extract from pomegranate, wherein the extract is prepared by aprocess comprising the steps of: (a) crushing and squeezing the wholefruits of pomegranate including inner and outer peels and the seeds toyield a juice component and an insoluble by-product component; and (b)separating the soluble juice component from the insoluble by-productcomponent and removing the insoluble by-product component therefrom toproduce the extract.
 19. The method of claim 18, wherein the stroke orthe heart attack is associated with artery arteriosclerotic disease. 20.The method of claim 18, wherein the artery arteriosclerotic disease iscarotid or coronary artery arteriosclerotic disease.
 21. The method ofclaim 18, wherein step (a) further comprises enzymatically treating thewhole fruits of pomegranate including inner and outer peels and theseeds.
 22. The method of claim 1, 10 or 18, wherein the process ofpreparing the extract further comprises the steps of: (c) resuspendingthe insoluble by-product component in an aqueous medium; (d) crushing,squeezing, and mixing the resuspended by-product component to yield asoluble portion and an insoluble portion; and (e) separating the solubleportion from the insoluble portion and removing the insoluble portiontherefrom to produce the extract.
 23. The method of claim 23, whereinstep (a) further comprises enzymatically treating the whole fruits ofpomegranate including inner and outer peels and the seeds.
 24. Themethod of claim 22, wherein the extract is prepared by a process furthercomprising the step of combining the extract of step (b) with theextract of step (e) to produce the extract.